Hybrid protein C

ABSTRACT

Human protein C molecules are modified to provide increased resistance to inactivation by human plasma factors while retaining substantially the biological activity of human protein C. The modifications are generally to the heavy chain of protein C, which chain may be substituted with a protein C heavy chain of non-human origin, such as bovine, yielding a chimeric protein C molecule. The human protein C heavy chain may also be modified to be human-like, in that at least one amino acid from a non-human sequence may be substituted for the corresponding residue(s) of the human sequence, thereby allowing the molecule to retain substantially human characteristics yet having increased resistance to inactivation. Also included are methods for producing the modified protein C molecules and pharmaceutical compositions thereof. The modified molecules, having an increased half-life in human plasma, are particularly useful for treating coagulation-related disorders, such as protein C deficiency or thrombosis, or for promoting fibrinolysis in a patient.

RELATED APPLICATIONS

This application is a divisional of Ser. No. 08/328,295, filed Oct. 24,1994 (pending), which is a divisional of Ser. No. 08/126,440, filed Sep.23, 1993 (issued as U.S. Pat. No. 5,358,932), which is a continuation ofSer. No. 07/634,988, filed Dec. 27, 1990 (abandoned), which is acontinuation-in-part of Ser. No. 07/515,378, filed Apr. 27, 1990(abandoned), which is a continuation-in-part of Ser. No. 07/458,856,filed Dec. 29, 1989 (abandoned), which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates generally to blood proteins and, moreparticularly, to compositions of human-like protein C molecules havingincreased resistance to inactivation by human plasma and thus improvedpharmacokinetics in vivo, and methods for producing such compositions.

BACKGROUND OF THE INVENTION

Protein C in its activated form plays an important role in regulatingblood coagulation. The activated protein C, a serine protease,inactivates coagulation Factors Va and VIIIa by limited proteolysis. Thecoagulation cascade initiated by tissue injury, for example, isprevented from proceeding in an unimpeded chain-reaction beyond the areaof injury by protein C.

Protein C is synthesized in the liver as a single chain precursorpolypeptide which is subsequently processed to a light chain of about155 amino acids (M_(r) =21,000) and a heavy chain of 262 amino acids(M_(r) =40,000). The heavy and light chains circulate in the blood as atwo-chain inactive protein, or zymogen, held together by a disulfidebond. When a 12 amino acid residue is cleaved from the amino-terminus ofthe heavy chain portion of the zymogen in a reaction mediated bythrombin, the protein becomes activated. Another blood protein, referredto as "protein S," is believed to somehow accelerate the proteinC-catalyzed proteolysis of Factor Va.

Protein C has also been implicated in the action of tissue-typeplasminogen activator (Kisiel and Fujikawa, Behring Inst. Mitt.73:29-42, 1983). Infusion of bovine activated protein C (APC) into dogsresults in increased plasminogen activator activity (Comp and Esmon, J.Clin. Invest. 68:1221-1228, 1981). Other studies (Sakata et al., Proc.Natl. Acad. Sci. USA 82:1121-1125, 1985) have shown that addition of APCto cultured endothelial cells leads to a rapid, dose-dependent increasein fibrinolytic activity in the conditioned media, reflecting increasesin the activity of both urokinase-related and tissue-type plasminogenactivators. APC treatment also results in a dose-dependent decrease inanti-activator activity. In addition, studies with monoclonal antibodiesagainst endogenous APC (Snow et al. FASEB Abstracts, 1988) implicate APCin maintaining patency of arteries during fibrinolysis and limiting theextent of tissue infarct.

Experimental evidence indicates that activated protein C may beclinically useful in the treatment of thrombosis. Several studies withbaboon models of thrombosis have indicated that APC in low doses will beeffective in prevention of fibrin deposition, platelet deposition andloss of circulation (Gruber et al., Hemostasis and Thrombosis 374a:abstract 1353, 1987; Widrow et al., Fibrinolysis 2 suppl. 1: abstract 7,1988; Griffin et al., Thromb. Haemostasis 62: abstract 1512, 1989). Theuse of APC bypasses the need for in vivo activation of protein C, thusproviding a faster acting therapeutic agent.

In addition, exogenous activated protein C has been shown to prevent thecoagulopathic and lethal effects of gram negative septicemia (Taylor etal., J. Clin. Invest, 19:918-925, 1987). Data obtained from studies withbaboons suggest that activated protein C plays a natural role inprotecting against septicemia.

Protein C may be purified from clotting factor concentrates (Marlar etal., Blood 59:1067-1072, 1982) or from plasma (Kisiel, J. Clin. Invest.64:761-769, 1979) and activated in vitro, but the resulting product maybe contaminated with such infectious agents as hepatitis virus,cytomegalovirus, or human immunodeficiency virus (HIV).

More recently, methods for producing activated protein C throughrecombinant DNA technology have been described. Foster et al. (publishedEuropean Patent Application EP 215,548) disclose the production ofactivated protein C through the use of cultured mammalian cellstransfected with a protein C DNA sequence from which the coding sequencefor the activation peptide has been deleted. Foster et al. (EP 266,190)disclose the production of recombinant activated protein C using a DNAsequence encoding an APC precursor with a modified cleavage site.

Moreover, native human activated protein C (either plasma-derived orrecombinant) has a relatively short half-life when administered in vivo(about twenty minutes), necessitating the inconvenience of large dosesor frequent administration.

Despite the advances in activated protein C production made possible bythe use of genetic engineering, yields remain low and the protein issubject to degradation and/or inactivation during the productionprocess. Thus, there remains a need in the art for methods that enablethe production of active activated protein C at higher levels andespecially the production of molecules which have a substantiallyincreased half-life in vivo. Quite surprisingly, the present inventionfulfills these and other related needs.

SUMMARY OF THE INVENTION

Novel compositions comprising protein C having a light chain and ahuman-like heavy chain are provided. The protein C may be in either itszymogen or activated form. The activated protein C which has ahuman-like heavy chain will generally be more resistant to inactivationby human plasma factors, such as human alpha-1-antitrypsin, whencompared to unaltered, naturally occurring protein C. The compositionsare particularly useful in methods for treating patients when they areformulated into pharmaceutical compositions, where they may be givenprophylactically or therapeutically to individuals suffering from avariety of disease states. Among the medical indications are protein Cdeficiencies, which may be an inherited disorder or an acquiredcondition. Other acquired disease states which may be treated with thenovel protein C molecules described herein include, e.g., deep veinthrombosis, pulmonary embolism, stroke, and myocardial infarction. Inthe latter, protein C may be administered with tissue plasminogenactivator to enhance in vivo fibrinolysis, and may be given after theoccluding coronary thrombus is dissolved to prevent reocclusion.

Typically, the light chain of the novel protein C molecule will besubstantially human, and the human-like heavy chain will comprise atleast about 200 amino acids from a human protein C heavy chain sequence,which sequence is generally about 262 residues in the zymogen form andgenerally about 250 residues in the activated form. In certain preferredembodiments the non-human residues of the human-like heavy chainoriginate from bovine sequences. The bovine heavy chain sequencesubstitutions for the human heavy chain sequence regions includesubstituting Gln-Glu-Ala-Gly-Trp for human amino acid sequenceLys-Met-Thr-Arg-Arg; bovine sequence Arg-Asp-Glu-Thr for human sequenceHis-Ser-Ser-Arg-Glu-Lys-Glu-Ala; bovine sequenceTyr-Asn-Ala-Cys-Val-His-Ala-Met-Glu-Asn-Lys is substituted for humanamino acid sequence His-Asn-Glu-Cys-Ser-Glu-Val-Met-Ser-Asn-Met; and thebovine region Lys-Ala-Gln-Glu-Ala-Pro-Leu-Glu-Ser-Gln-Pro-Val issubstituted for human heavy chain regionArg-Asp-Lys-Glu-Ala-Pro-Gln-Lys-Ser-Trp-Ala-Pro. Of course, it will beunderstood that certain minor substitutions, insertions or deletions maybe made in the human heavy chain framework or non-human regions, so longas the protein C molecule retains biological activity. Desirably, suchprotein C analogs will have, e.g., increased resistance to inactivationby human plasma and thus a longer plasma half-life or increasedbiological activity.

In another embodiment the invention concerns a recombinant chimericprotein C molecule having light and heavy chain polypeptides, where thelight chain is substantially human and the heavy chain is substantiallythat of a mammal other than human, preferably bovine. This form ofprotein C will have substantially the activity of human protein C, butwill be more resistant to inactivation by human plasma factors than thenaturally occurring human protein C. The sequence of the heavy chain ofthis embodiment may be substantially homologous to the bovine heavychain sequence of FIG. 8; a preferred composition has an amino terminalamino acid of the human activated heavy chain (Leu), while the remainderof the heavy chain is substantially bovine.

In another aspect the invention relates to a polynucleotide moleculecomprising four operatively linked sequence coding regions which encode,respectively, a pre-pro peptide and a gla domain of a vitaminK-dependent plasma protein, a gla domain-less human protein C lightchain, a peptide comprising one or more cleavage sites, and a human-likeprotein C heavy chain. The protein C molecule expressed by thispolynucleotide is biologically active, that is, in its activated form itis capable of inactivating human plasma Factors Va or VIIIa, yet itselfhas increased resistance to inactivation by human factors such asalpha-1-antitrypsin. To express the protein C molecule the nucleotidesequences are transfected into mammalian cell lines, such as BHK, BHK570 and 293, and may be cotransfected with sequences which encodeendopeptidases reactive at the cleavage sites, such as the KEX2 gene ofSaccharomyces cerevisiae.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B illustrates the nucleotide sequence of a human protein C cDNAand the inferred amino acid sequence of the protein. Negative numbersrefer to the pre-pro peptide. Positive numbers refer to the sequence ofthe mature zymogen. Diamonds indicate consensus N-linked glycosylationsites. The arrow indicates the junction between the activation peptideand the activated protein C heavy chain.

FIG. 2 depicts the protein C expression vector p594. Symbols used are0-1, the adenovirus 5 0-1 map unit sequence; E, the SV40 enhancer; MLP,the adenovirus 2 major late promoter; L1-3, the adenovirus 2 tripartiteleader; 5',5' splice site; 3',3' splice site; p(A), the SV40 latepolyadenylation signal.

FIG. 3 illustrates the construction of the protein C expression vectorPC962/ZMB-4.

FIG. 4 illustrates the construction of the vector ZMB-3.

FIG. 5 illustrates the results of chromogenic activity assays on human,bovine, and hybrid protein C molecules. Data for each protein arenormalized to 100% activity in the absence of alpha-1-antitrypsin.

FIG. 6 illustrates the inactivation of human and hybrid protein Cmolecules by human plasma. Results are normalized for each protein.

FIG. 7 illustrates the time course of inactivation of human and hybridprotein C molecules by human plasma. Results are normalized for eachprotein.

FIG. 8 shows a comparison of the amino acid sequences of the heavychains of human and bovine protein C. Each sequence is numbered from thefirst amino acid of the respective heavy chain. The arrow indicates thejunction between the activation peptide and the activated protein Cheavy chain. Within the bovine sequence, (.) indicates the presence ofthe same amino acid residue as in the human sequence, and (-) indicatesa gap introduced to maximize sequence alignment.

FIG. 9 illustrates the inactivation of human (wt 962), hybrid (LMH),mutant PC2451, PC2452 and PC3044 protein C molecules by α-1-antitrypsin.Results are normalized for each protein.

FIG. 10 illustrates a time course over 300 minutes for the inactivationof activated human (wt 962), hybrid (LMH), mutant PC2451, PC2452 andPC3044 protein C molecules by human plasma. Results are normalized foreach protein.

FIG. 11 illustrates a time course over 60 minutes for the inactivationof activated human (wt 962), hybrid (LMH), and mutant PC2451 protein Cmolecules by human plasma. Results are normalized for each protein.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Novel compositions of protein C are provided which are suitable foradministration to humans. Because of the pivotal role protein C plays inthe coagulation cascade, acting in its activated form as ananticoagulant, it has a variety of important therapeutic applications.The novel compositions described herein provide a possibility ofachieving an extended half-life and stability in vivo not achievablewith prior compositions of protein C purified from human plasma orproduced by recombinant means.

In one aspect of the invention, the composition comprises a hybrid, orchimeric, protein C molecule where the amino acid sequence of the lightchain is substantially human and the sequence of the heavy chain issubstantially that of a mammal other than human, such as bovine. It mayalso be desirable or convenient that the amino-terminal amino acid ofthe activated heavy chain be from the human sequence, which is typicallyleucine (Leu). The remainder of the heavy chain may be entirely of thebovine heavy chain sequence. It should be understood that referenceherein to "protein C" is meant to include the zymogen and activatedforms, unless otherwise specified. Protein C zymogen includes anactivation peptide at the amino terminus of the heavy chain. Theactivation peptide may be the native human activation peptide, thenative bovine activation peptide, or a modified activation peptide asdisclosed herein.

In alternative embodiments, protein C molecules are produced which arehuman-like in nature, and thus possess generally less immunogenicitythan a chimeric molecule. Short sequences, including but not limited tosingle amino acids, of the human heavy chain (as shown in FIG. 8, forexample) may be replaced with the corresponding heavy chain sequencesfrom protein C of a mammal other than human, conveniently bovinesequences. The object is to achieve protein C molecules which aresubstantially human and, when activated, will have a substantiallylonger half-life in human blood, thereby necessitating less frequentadministration and/or smaller dosages. As used herein, the term"human-like heavy chain" is meant to refer to a protein C heavy chainwhich is substantially homologous to an authentic human heavy chain,i.e., at least about 75%, and preferably about 85-95% identical,particularly in those regions which are relatively conserved amongspecies, and containing at least one amino acid substitution. Ingeneral, it is preferred to retain the interaction of protein C withplasminogen activator inhibitor-1 by retaining the basic amino acidresidues at positions 191-193.

The hybrid or human-like protein C is produced by cultured mammaliancells transfected with genes which encode the hybrid or human-likemolecules. The cells are transfected with an expression vectorcomprising a promoter operably linked to a DNA encoding the protein Cmolecule. The transfected cells are cultured in a medium which permitsthe expression of the protein, and protein C is then isolated from themedium. The protein C may be produced in its activated or zymogen form.If it is produced in its activated form the medium should be prepared soas to contain a minimal amount of serum, or to be serum-free, asdescribed in commonly owned copending application Ser. No. 07/392,861,incorporated herein by reference.

Cloned DNA sequences encoding human protein C have been described(Foster and Davie, Proc. Natl. Acad. Sci. USA 81:4766-4770, 1984; Fosteret al., Proc. Natl. Acad. Sci. USA 82:4673-4677, 1985 and Bang et al.,U.S. Pat. No. 4,775,624, each incorporated herein by reference). A cDNAencoding bovine protein C has been described by Long et al., Proc. Natl.Acad. Sci. USA 81:5563-5656, 1984, incorporated herein by reference. Ingeneral, cDNA sequences are preferred for use within the presentinvention due to their lack of intervening sequences which can lead toaberrant RNA processing and reduced expression levels. ComplementaryDNAs encoding protein C may be obtained from libraries prepared fromliver cells of various mammalian species according to standardlaboratory procedures. Using probes from bovine or human cDNA, one mayidentify and clone the DNA encoding protein C of other mammalianspecies. It will be understood, however, that suitable DNA sequences canalso be obtained from genomic clones or can be synthesized de novoaccording to conventional procedures. If partial clones are obtained, itis necessary to join them in proper reading frame to produce a fulllength clone, using such techniques as endonuclease cleavage, ligation,and loop-out mutagenesis.

For example, to clone bovine cDNA encoding the protein C heavy chain, ahuman protein C cDNA fragment may be used to probe a bovine liver cDNAlibrary. The human protein C cDNA fragment may be prepared from humanliver DNA, for example, using conventional methods. Alternatively,probes may be designed based on the published bovine protein C cDNAsequence (Long et al., Proc. Natl. Acad. Sci. USA 81:5653-5656, 1984). Ahybrid protein C coding sequence may be constructed by joining the humanlight chain cDNA to the non-human fragment (e.g., bovine) in properreading frame to produce a full length protein, using cleavage withappropriate restriction endonucleases, ligation, syntheticoligonucleotides and loop-out mutagenesis.

In alternative embodiments, the entire human protein C coding region maybe cloned and selected modifications made to the heavy chain sequence toincrease the half-life and stability of the molecule in human plasma bymaking it resistant to inhibition or inactivation by factors in humanplasma. The modifications will be directed to areas of the heavy chainwhere the amino acid sequence differs substantially among species. Thus,the species-specificity of human alpha-1-antitrypsin for the human heavychain is used to make site(s) typically recognized byalpha-1-antitrypsin in the heavy chain less susceptible to recognitionand thereby inhibit the rate at which the activity of protein C isdegraded. With the bovine heavy chain sequence, for example, thefollowing sequences have been identified as sites which may besubstituted for corresponding human heavy chain regions (numberingaccording to Foster et al., Proc. Natl. Acad. Sci. USA 81:4766-4770,1984 and as shown in FIG. 8):

(1) a bovine heavy chain amino acid sequence Gln-Glu-Ala-Gly-Trp (heavychain amino acids 19-23), is substituted for human amino acid sequenceLys-Met-Thr-Arg-Arg (heavy chain amino acids 17-21);

(2) bovine heavy chain amino acid sequence Arg-Asp-Glu-Thr (amino acids148-151), is substituted for human amino acid sequenceHis-Ser-Ser-Arg-Glu-Lys-Glu-Ala (amino acids 146-153);

(3) bovine heavy chain amino acid sequenceTyr-Asn-Ala-Cys-Val-His-Ala-Met-Glu-Asn-Lys (amino acids 169-179), issubstituted for human amino acid sequenceHis-Asn-Glu-Cys-Ser-Glu-Val-Met-Ser-Asn-Met (amino acids 171-181); and

(4) bovine heavy chain amino acid sequenceLys-Ala-Gln-Glu-Ala-Pro-Leu-Glu-Ser-Gln-Pro-Val (amino acids 249-260) issubstituted for human heavy chain residuesArg-Asp-Lys-Glu-Ala-Pro-Gln-Lys-Ser-Trp-Ala-Pro (amino acids 251-262).

It will be understood that preferably as few sequence modifications aspossible are made to provide an increased resistance to inactivation byhuman plasma factors. Desirably, the region of substitution will be assmall as possible, such that most preferred will be a single heavy chainamino acid substitution of one species for a corresponding amino acid ofthe human sequence. Combinations of sequence region substitutions mayalso be employed. The increased resistance of a molecule to degradationmay be readily assayed with well known procedures, such as resistance toalpha-1-antitrypsin or human plasma, as described below. It is importantthat any such substitutions not substantially decrease the biologicalactivity of protein C. By "biological activity" is meant a function orset of functions performed by activated human protein C in a biologicalcontext (i.e., in an organism or an in vitro model thereof). Biologicalactivities of proteins may be divided into catalytic and effectoractivities. Catalytic activities of vitamin K-dependent plasma proteins,such as protein C, generally involve specific proteolytic cleavages ofother plasma proteins, resulting in activation or deactivation of thesubstrates. Effector activities include specific binding of thebiologically active molecules to calcium, phospholipid or other smallmolecules, to macromolecules, such as proteins, or to cells. Effectoractivity frequently augments, or is essential to, catalytic activityunder physiological conditions. For protein C, biological activity ischaracterized by the anticoagulant properties of the activated protein.Activated protein C inactivates Factor Va and Factor VIIIa in thepresence of acidic phospholipid and calcium. Protein S appears to beinvolved in the regulation of this function (Walker, ibid.). Thecatalytic activities of protein C are believed to reside primarily inthe heavy chain. These activities may be readily assayed using wellknown procedures.

To produce recombinant activated protein C directly, the cloned DNAsequence is modified to delete or replace that portion encoding theactivation peptide. The resulting DNA sequence will encode a pre-propeptide, the light chain of protein C, a cleavage site and the heavychain of activated protein C. The DNA sequence may further encode aspacer peptide between the light and heavy chains.

In one embodiment, the resultant sequence will encode the light andheavy chains of protein C joined by the sequence Lys-Arg. As usedherein, the light chain of activated protein C is understood to compriseamino acids 1-149 of the sequence disclosed in FIGS. 1A-B or sequencessubstantially homologous thereto, or such sequences having C-terminalextensions, generally extensions of one to about six amino acids. Theheavy chain of activated protein C is understood not to include theactivation peptide (i.e. to begin at amino acid number 170, leucine, asshown in FIG. 1A-B in the case of human activated protein C).

In a preferred embodiment, the DNA sequence is further modified toinclude one or more novel cleavage sites between the light and heavychains. The cleavage site may be in the form of the amino acid sequence(R₁)_(n) --R₂ --R₃ --R₄, wherein R₁ through R₄ are lysine (Lys) orarginine (Arg) and n is an integer between 0 and 3. Particularlypreferred sequences include Arg-Arg-Lys-Arg, Lys-Arg-Lys-Arg andLys-Lys-Arg. Alternatively, the cleavage site may be of the form R₁ --R₂--R₃ --R₄ --X--R₅ --R₆ --R₇ --R₈, wherein each of R₁ through R₈ is Lysor Arg and X is a peptide bond or a spacer peptide of 1 to 12 aminoacids. Spacer peptides useful in this regard include the amino acidsequences Asp-Thr-Glu-Asp-Gln-Glu-Asp-Gln-Val-Asp-Pro,Asp-Thr-Glu-Asp-Gln-Glu-Asp-Gln, Asp-Thr-Asp-Gln, Asp-Gln,Asn-Ile-Leu-Asn, and the native protein C activation peptide having theamino acid sequence Asp-Thr-Glu-Asp-Gln-Glu-Asp-Gln-Val-Asp-Pro-Arg. Athird group of cleavage site modifications includes the substitution ofamino acid residue 154 (His) of native human protein C with an aminoacid residue selected from the group consisting of Lys, Arg and Leu togive a cleavage site sequence of the general formula Y--Z--R₁ --R₂,wherein Y is Lys, Arg or Leu; R₁ and R₂ are Lys or Arg; and Z is anamino acid other than Lys or Arg, preferably Leu. Representativecleavage-site mutants which may be useful in the present invention areshown below in Table I. Cleavage sites 829, 1058, 1645, 1880, 1953,1954, 1962, 2043, 2155 and 2274 are useful in directly producingactivated protein C.

                                      TABLE I                                     __________________________________________________________________________    Amino Acid Sequences of Cleavage-Site Mutants                                 __________________________________________________________________________     ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                      ##STR7##                                                                      ##STR8##                                                                      ##STR9##                                                                      ##STR10##                                                                     ##STR11##                                                                     ##STR12##                                                                    __________________________________________________________________________

Modification of the DNA sequence may be obtained by site-specificmutagenesis. Techniques for site-specific mutagenesis are well known inthe art and are described by, for example, Zoller and Smith (DNA3:479-488, 1984). Alternatively, the wild-type protein C sequence may beenzymatically cleaved to remove the native activation peptide sequence,and the sequences encoding the heavy and light chains joined to asynthesized DNA sequence encoding one of the cleavage sites describedabove.

As will be understood by those skilled in the art, variants and analogsof protein C may also be produced in the context of the compositions andmethods of the present invention. Variants and analogs of protein Cinclude those containing minor amino acid changes, such as those due togenetic polymorphism, and those in which amino acids have been inserted,deleted and/or substituted without substantially decreasing thebiological activity of the protein. Protein C analogs further includeproteins that have the protein C amino-terminal portion (gla domain)substituted with a gla domain of one of the vitamin-K dependent plasmaproteins Factor VII, Factor IX, Factor X, prothrombin or protein S. Thegla domain spans approximately 35-45 amino acid residues at the aminotermini of these proteins, with a C-terminal boundary generallycorresponding to an exon-intron boundary in the respective gene. The gladomain of human protein C extends from amino acid number 1 of the maturelight chain to approximately amino acid number 37.

DNA sequences for use within the present invention will encode a pre-propeptide at the amino-terminus of the hybrid protein C molecule to obtainproper post-translational processing (e.g. gamma-carboxylation ofglutamic acid residues) and secretion from the host cell. The pre-propeptide may be that of protein C or another vitamin K-dependent plasmaprotein, such as Factor VII, Factor IX, Factor X, prothrombin or proteinS. It is generally preferred that the pre-pro peptide and gla domain beobtained from the same protein.

The DNA sequence encoding the hybrid protein C is inserted into asuitable expression vector, which vector is then used to transfectcultured mammalian cells. Expression vectors for use in carrying out thepresent invention will comprise a promoter capable of directing thetranscription of a cloned gene or cDNA. Preferred promoters includeviral promoters and cellular promoters. Viral promoters include the SV40promoter (Subramani et al., Mol. Cell. Biol. 1:854-864, 1981) and theCMV promoter (Boshart et al., Cell 41:521-530, 1985). A particularlypreferred viral promoter is the major late promoter from adenovirus 2(Kaufman and Sharp, Mol. Cell. Biol. 2:1304-1319, 1982). Cellularpromoters include the mouse kappa gene promoter (Bergman et al., Proc.Natl. Acad. Sci. USA 81:7041-7045, 1983) and the mouse V_(H) promoter(Loh et al., Cell 33:85-93, 1983). A particularly preferred cellularpromoter is the mouse metallothionein-I promoter (Palmiter et al.,Science 222:809-814, 1983). Expression vectors may also contain a set ofRNA splice sites located downstream from the promoter and upstream fromthe insertion site for the protein C sequence itself. Preferred RNAsplice sites may be obtained from adenovirus and/or immunoglobulingenes. Also contained in the expression vectors is a polyadenylationsignal located downstream of the insertion site. Particularly preferredpolyadenylation signals include the early or late polyadenylation signalfrom SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal fromthe adenovirus 5 Elb region, the human growth hormone gene terminator(DeNoto et al. Nuc. Acids Res. 9:3719-3730, 1981) or the polyadenylationsignal from the human protein C gene or the bovine protein C gene. Theexpression vectors may also include a noncoding viral leader sequence,such as the adenovirus 2 tripartite leader, located between the promoterand the RNA splice sites; and enhancer sequences, such as the SV40enhancer and the sequences encoding the adenovirus VA RNAS.

Cloned DNA sequences are introduced into cultured mammalian cells by,for example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725-732, 1978; Corsaro and Pearson, Somatic Cell Genetics7:603-616, 1981; Graham and Van der Eb, Virology 52d:456-467, 1973) orelectroporation (Neumann et al., EMBO J. 1:841-845, 1982). To identifyand select cells that express the exogenous DNA, a gene that confers aselectable phenotype (a selectable marker) is generally introduced intocells along with the gene or cDNA of interest. Preferred selectablemarkers include genes that confer resistance to drugs such as neomycin,hygromycin, and methotrexate. The selectable marker may be anamplifiable selectable marker. A preferred amplifiable selectable markeris a dihydrofolate reductase (DHFR) sequence. Selectable markers arereviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers,Stoneham, Mass., incorporated herein by reference). The choice ofselectable markers is well within the level of ordinary skill in theart.

Selectable markers may be introduced into the cell on a separate plasmidat the same time as the gene of interest, or they may be introduced onthe same plasmid. If on the same plasmid, the selectable marker and thegene of interest may be under the control of different promoters or thesame promoter, the latter arrangement producing a dicistronic message.Constructs of this type are known in the art (for example, Levinson andSimonsen, U.S. Pat. No. 4,713,339). It may also be advantageous to addadditional DNA, known as "carrier DNA," to the mixture that isintroduced into the cells.

After the cells have taken up the DNA, they are grown in an appropriategrowth medium, typically 1-2 days, to begin expressing the gene ofinterest. As used herein the term "appropriate growth medium" means amedium containing nutrients and other components required for the growthof cells and the expression of the protein C gene. Media generallyinclude a carbon source, a nitrogen source, essential amino acids,essential sugars, vitamins, salts, phospholipids, protein and growthfactors. For production of gamma-carboxylated protein C, the medium willcontain vitamin K, preferably at a concentration of about 0.1 μg/ml toabout 5 μg/ml. Drug selection is then applied to select for the growthof cells that are expressing the selectable marker in a stable fashion.For cells that have been transfected with an amplifiable selectablemarker the drug concentration may be increased to select for anincreased copy number of the cloned sequences, thereby increasingexpression levels. Clones of stably transfected cells are then screenedfor expression of protein C.

Preferred mammalian cell lines for use in the present invention includethe COS-1 (ATCC CRL 1650), baby hamster kidney (BHK) and 293 (ATCC CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) cell lines.Preferred BHK cell lines include the tk⁻ ts13 BHK cell line (Waechterand Baserga, Proc. Natl. Acad. Sci. USA 79:1106-1110, 1982, incorporatedherein by reference), hereinafter referred to as BHK 570 cells. The BHK570 cell line has been deposited with the American Type CultureCollection, 12301 Parklawn Dr., Rockville, Md. 20852 prior to the filingof this patent application under ATCC accession number CRL 10314. A tk⁻ts13 BHK cell line is also available from the ATCC under accessionnumber CRL 1632. In addition, a number of other cell lines may be usedwithin the present invention, including Rat Hep I (ATCC CRL 1600), RatHep II (ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065),NCTC 1469 (ATCC CCL 9.1), CHO (ATCC CCL 61) and DUKX cells (Urlaub andChasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980).

Processing of activated protein C precursors by cleavage after a Lys-Argdipeptide between the light and heavy chains may be enhanced byintroducing the S. cerevisiae KEX2 gene into the host cell, as describedin published European patent application EP 319,944. The KEX2 geneencodes an endopeptidase that cleaves after a dibasic amino acidsequence (Fuller et al., in Leive, ed., Microbiology 1986, 273-278). Acultured mammalian cell line stably transfected with this gene is thususeful for expressing activated protein C.

Protein C produced according to the present invention may be purified byaffinity chromatography on an anti-protein C antibody column. The use ofcalcium-dependent monoclonal antibodies, as described by Wakabayashi etal. (J. Biol. Chem. 261:11097-11108, 1986), is particularly preferred.Additional purification may be achieved by conventional chemicalpurification means, such as liquid chromatography. Other methods ofpurification, including barium citrate precipitation, are known in theart, and may be applied to the purification of the novel protein Cdescribed herein (see, generally, Scopes, R., Protein Purification,Springer-Verlag, N.Y., 1982). Substantially pure protein C of at leastabout 90 to 95% homogeneity is preferred, and 98 to 99% or morehomogeneity most preferred, for pharmaceutical uses. Once purified,partially or to homogeneity as desired, the protein C may then be usedtherapeutically.

The protein C molecules of the present invention and pharmaceuticalcompositions thereof are particularly useful for administration tohumans to treat a variety of conditions involving intravascularcoagulation. For instance, although deep vein thrombosis and pulmonaryembolism can be treated with conventional anticoagulants, the protein Cdescribed herein may be used to prevent the occurrence of thromboemboliccomplications in identified high risk patients, such as those undergoingsurgery or those with congestive heart failure. Since activated proteinC is more selective than heparin, being active in the body generallywhen and where thrombin is generated and fibrin thrombi are formed,protein C will be more effective and less likely to cause bleedingcomplications than heparin when used prophylactically for the preventionof deep vein thrombosis. The dose of protein C for prevention of deepvein thrombosis is in the range of about 100 μg to 100 mg/day,preferably 1 to 10 mg/day, and administration should begin at leastabout 6 hours prior to surgery and continue at least until the patientbecomes ambulatory. In established deep vein thrombosis and/or pulmonaryembolism, the dose of protein C ranges from about 100 μg to 100 mg as aloading dose followed by maintenance doses ranging from 3 to 300 mg/day.Because of the lower likelihood of bleeding complications from protein Cinfusions, protein C can replace or lower the dose of heparin during orafter surgery in conjunction with thrombectomies or embolectomies.

The protein C compositions of the present invention will also havesubstantial utility in the prevention of cardiogenic emboli and in thetreatment of thrombotic strokes. Because of its low potential forcausing bleeding complications and its selectivity, protein C can begiven to stroke victims and may prevent the extension of the occludingarterial thrombus. The amount of protein C administered will vary witheach patient depending on the nature and severity of the stroke, butdoses will generally be in the range of those suggested below.

Pharmaceutical compositions of activated protein C provided herein willbe a useful treatment in acute myocardial infarction because of theability of activated protein C to enhance in vivo fibrinolysis.Activated protein C can be given with tissue plasminogen activator orstreptokinase during the acute phases of the myocardial infarction.After the occluding coronary thrombus is dissolved, activated protein Ccan be given for subsequent days or weeks to prevent coronaryreocclusion. In acute myocardial infarction, the patient is given aloading dose of at least about 1-500 mg of activated protein C, followedby maintenance doses of 1-100 mg/day.

The protein C of the present invention is useful in the treatment ofdisseminated intravascular coagulation (DIC), in either its zymogen oractivated form. Patients with DIC characteristically have widespreadmicrocirculatory thrombi and often severe bleeding problems which resultfrom consumption of essential clotting factors. Because of itsselectivity, protein C will not aggravate the bleeding problemsassociated with DIC, as do conventional anticoagulants, but will retardor inhibit the formation of additional microvascular fibrin deposits.

As the novel protein C molecules provided herein generally have a longerhalf-life than authentic human protein C, a significant use of thesecompositions is the treatment of people who have an inherited protein Cdeficiency. Such patients, who may be homozygous or heterozygous for thedeficiency, may suffer from severe thrombosis. They are presentlymaintained on Factor IX concentrates, which contain protein C. Fortreatment of the homozygous deficient individuals, assuming an averageblood plasma volume of about 3,000 ml and allowing for some diffusioninto the extravascular space, protein C of the invention may beadministered one or more times daily at levels of from 1-300 mg daily.Heterozygotes for protein C deficiency will generally require lowermaintenance doses than homozygotes.

The pharmaceutical compositions are intended for parenteral, topical,oral or local administration for prophylactic and/or therapeutictreatment. Preferably, the pharmaceutical compositions are administeredparenterally, i.e., intravenously, subcutaneously, or intramuscularly.Thus, this invention provides compositions for parenteral administrationwhich comprise a solution of the protein C molecules dissolved in anacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers may be used, e.g., water, buffered water, 0.4% saline, 0.3%glycine and the like. These compositions may be sterilized byconventional, well known sterilization techniques. The resulting aqueoussolutions may be packaged for use or filtered under aseptic conditionsand lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, etc. The concentration of protein C in these formulations canvary widely, i.e., from less than about 0.5%, usually at or at leastabout 1% to as much as 15 or 20% by weight and will be selectedprimarily by fluid volumes, viscosities, etc., in accordance with theparticular mode of administration selected.

Thus, a typical pharmaceutical composition for intravenous infusioncould be made up to contain 250 ml of sterile Ringer's solution, and 10mg of protein C. Actual methods for preparing parenterally administrablecompounds will be known or apparent to those skilled in the art and aredescribed in more detail in for example, Remington's PharmaceuticalScience, 16th ed., Mack Publishing Company, Easton, Pa. (1982), which isincorporated herein by reference.

The compositions containing the protein C molecules or a cocktailthereof can be administered for prophylactic and/or therapeutictreatments. In therapeutic applications, compositions are administeredto a patient already suffering from a disease, as described above, in anamount sufficient to cure or at least partially arrest the disease andits complications.

An amount adequate to accomplish this is defined as "therapeuticallyeffective dose." Amounts effective for this use will depend on theseverity of the disease or injury and the general state of the patient,but generally range from about 1 mg to about 300 mg of protein C perday, with dosages of from about 5 mg to about 25 mg of protein C per daybeing more commonly used. It must be kept in mind that the materials ofthe present invention may generally be employed in serious disease orinjury states, that is, life-threatening or potentially life threateningsituations. In such cases, in view of the minimization of extraneoussubstances and the prolonged half-life of protein C in human plasma madefeasible by this invention, it is possible and may be felt desireable bythe treating physician to administer substantial excesses of theseprotein C compositions.

In prophylactic applications, compositions containing the hybrid proteinC are administered to a patient susceptible to or otherwise at risk of adisease state or injury to enhance the patient's own anticoagulative orfibrinolytic capabilities. Such an amount is defined to be a"prophylactically effective dose." In this use, the precise amountsagain depend on the patient's state of health and general level ofendogenous protein C, but generally range from about 0.5 mg to about 250mg per 70 kilogram patient, especially about 1 mg to about 25 mg per 70kg. of body weight.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. For ambulatory patients requiring daily maintenance levels,the protein C may be administered by continuous infusion using aportable pump system, for example. In any event, the pharmaceuticalformulations should provide a quantity of protein C of this inventionsufficient to effectively treat the patient.

The following examples are offered by way of illustration of theinvention and not by limitation.

EXAMPLE I Construction of Human-Bovine Hybrid Protein C

This Example describes the construction of a hybrid protein C codingsequence encoding a hybrid protein C having a human Pre-Pro sequence, ahuman light chain, a human activation peptide and the first amino acidof the human activated protein C heavy chain, followed by the remainderof the bovine protein C heavy chain sequence. Following secretion fromthe host cell and activation, the protein comprises the human protein Clight chain which is disulfide bonded to a heavy chain containing thefirst amino acid (leucine) of the human heavy chain followed by thebovine heavy chain sequence from the second amino acid (valine). Thehybrid molecule is shown in the Examples which follow to have increasedresistance to inactivation by α-1-antitrypsin and other human plasmafactors.

A. Bovine Heavy Chain cDNA Cloning

Bovine cDNA encoding the protein C heavy chain was cloned from a bovineliver cDNA λgt11 library (obtained from Clontech, Palo Alto, Calif.94301) by probing the library with a random-primed human protein C cDNAfragment.

1. Preparation of Human Protein C cDNA Probe

A cDNA sequence coding for a portion of human protein C was prepared asdescribed by Foster and Davie (ibid.). Briefly, a λgt11 cDNA library wasprepared from human liver mRNA by conventional methods. Clones werescreened using an ¹²⁵ I-labeled affinity-purified antibody to humanprotein C, and phage were prepared from positive clones by the platelysate method (Maniatis et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, 1982, incorporated herein by reference), followed bybanding on a cesium chloride gradient. The cDNA inserts were removedusing Eco RI and were subcloned into plasmid pUC9 (Vieira and Messing,Gene 19:259-268, 1982). Restriction fragments were subcloned in phagevectors M13mp10 and M13mp11 (Messing, Meth. Enzymol. 101:20-77, 1983)and were sequenced by the dideoxy method (Sanger et al., Proc. Natl.Acad. Sci. USA 74:5464-5467, 1977). A clone that contained DNAcorresponding to the known partial sequence of human protein C (Kisiel,ibid., 1979) and encoded protein C beginning at amino acid 64 of thelight chain and extending through the heavy chain and into the 3'non-coding region was selected. This clone was designated λHC1375. Asecond cDNA clone coding for protein C from amino acid 24 was alsoidentified. The insert from the larger clone was subcloned into pUC9 andthe plasmid was designated pHCλ6L. This clone encodes a major portion ofprotein C, including the heavy chain coding region, termination codon,and 3' non-coding region.

The cDNA insert from λHC1375 was nick translated using α-³² P dNTP's andused to probe a human genomic library in phage λCharon 4A (Maniatis etal., Cell 15:687-702, 1978) using the plaque hybridization procedure ofBenton and Davis (Science 196:181-182, 1977) as modified by Woo (Meth.Enzymol. 68:381-395, 1979).

Positive clones were isolated and plaque-purified (Foster et al., Proc.Natl. Acad. Sci. USA 82:4673-4677, 1985, herein incorporated byreference). Phage DNA prepared from positive clones (Silhavy et al., inExperiments with Gene Fusion, Cold Spring Harbor Laboratory, 1984) andwas digested with Eco RI or Bgl II and the genomic inserts were purifiedand subcloned in pUC9. (Restriction fragments of the genomic insertswere subcloned into M13 vectors and sequenced to confirm their identityand establish the DNA sequence of the entire gene.)

The cDNA insert of pHCλ6L was nick translated and used to probe thephage λCharon 4A library. One genomic clone was identified thathybridized to probes made from the 5' and 3' ends of the cDNA. Thisphage clone was digested with Eco RI, and a 4.4 kb fragment,corresponding to the 5' end of the protein C gene, was subcloned intopUC9. The resultant recombinant plasmid was designated pHCR4.4. CompleteDNA sequence analysis revealed that the insert in pHCR4.4 included twoexons of 70 and 167 base pairs separated by an intron of 1263 bp. Thefirst exon encodes amino acids -42 to -19; the second encodes aminoacids -19 to 37. Sequence analysis confirmed the DNA sequence of theentire protein C gene.

A genomic fragment containing an exon corresponding to amino acids -42to -19 of the pre-pro peptide of protein C was isolated, nicktranslated, and used as a probe to screen a cDNA library constructed bythe technique of Gubler and Hoffman (Gene 25:263-269, 1983) using mRNAfrom Hep G2 cells. This cell line was derived from human heptocytes andwas previously shown to synthesize protein C (Fair and Bahnak, Blood 64:194-204, 1984). Ten positive clones comprising cDNA inserted into theEco RI site of phage λgt11 were isolated and screened with anoligonucleotide probe corresponding to the 5' non-coding region of theprotein C gene. One clone was also positive with this probe and itsentire nucleotide sequence was determined. The cDNA contained 70 bp of5' untranslated sequence, the entire coding sequence for humanpre-pro-protein C, and the entire 3' non-coding region corresponding tothe second polyadenylation site. The cDNA sequence and the encoded aminoacid sequence are shown in FIGS. 1A-B.

The protein C cDNA was isolated as an Eco RI fragment and cloned intothe vector pDX (Hagen et al., U.S. Pat. No. 4,784,950, incorporatedherein by reference) as disclosed in published European PatentApplication EP 266,190, incorporated herein by reference.

Recombinant plasmids were screened by restriction analysis to identifythose having the protein C insert in the correct orientation withrespect to the promoter elements, and plasmid DNA (designated pDX/PC)was prepared from a correct clone. Because the cDNA insert in pDX/PCcontained an ATG codon in the 5' non-coding region,oligonucleotide-directed deletion mutagenesis was performed on the cDNAto remove the three base pairs. The resulting vector, designated p594,contained the protein C cDNA operably linked to the adenovirus 2 majorlate promoter (FIG. 2). This vector also contained the adenovirus 5origin of replication (0-1 map units sequence), the SV40 enhancer, theadenovirus 2 tripartite leader, a set of RNA splice sites, an SV40polyadenylation signal and a dihydrofolate reductase gene as aselectable marker.

2. Isolation of Bovine cDNA Clone

Using a random-primed 1.7 kb Eco RI fragment from p594 containing thehuman protein C cDNA, a bovine liver cDNA λgt11 library was probed forprotein C cDNA. A bovine clone was identified and recovered as an Eco RIfragment and was cloned into pUC9. The resultant plasmid was cut withTaq I and Eco RI, and the fragment encoding the protein C heavy chainwas recovered.

B. Preparation of Human Protein C Light Chain cDNA

To obtain a human light chain cDNA for ultimately joining to the bovineTaq I-Eco RI fragment, an appropriate restriction fragment was preparedfrom a DNA sequence (designated PC962) which encoded the human proteinC. The PC962 DNA was generated from p594, described above, and containeda DNA sequence encoding two additional arginine residues at the junctionbetween the light chain and the activation peptide of protein C (TableI). The cloned human cDNA in p594 was altered by site-specificmutagenesis (essentially as described by Zoller and Smith, DNA 3:479-488(1984)), using the mutagenic oligonucleotide ZC962 (5' AGT CAC CTG AGAAGA AAA CGA GAC A 3') and oligonucleotide ZC550 (5' TCC CAG TCA CGA CGT3'). Plasmid p594 was digested with Sst I, the approximately 840 bpfragment was cloned into M13mp11, and single-stranded template DNA wasisolated. Following mutagenesis, a correct clone was identified bysequencing. Replicative form DNA was isolated and digested with Sst I toisolate the mutagenized fragment. This fragment was joined with SstI-cut p594 in a two-part ligation. Clones having the Sst I fragmentinserted in the desired orientation were identified by restrictionenzyme mapping. The resulting expression vector was designatedpDX/PC962.

A second expression vector, designated PC229/962, was constructed byinserting the PC962 cDNA into plasmid Zem229. Zem229 is a pUC18-basedexpression vector containing a unique Bam HI site for insertion offoreign DNA between the mouse metallothionein-I promoter and SV40transcription terminator. Zem229 also contains an expression unit of theSV40 early promoter, mouse dihydrofolate reductase gene, and SV40terminator. An Eco RI fragment containing the PC962 cDNA from pDX/PC962was joined, via Eco RI-Bam HI synthetic oligonucleotide adapters, toZem229, which had been cut with Bam HI and treated with phosphatase. Theresulting vector, designated PC229/962, is illustrated in FIG. 3.

Expression vector PC962/ZMB-4 was constructed from Zem229, pDX (Hagen etal., U.S. Pat. No. 4,784,950, incorporated herein by reference) and thePC962 DNA sequence.

Zem229 was modified to convert the Bam HI cloning site to an Eco RIsite. The plasmid was first modified to delete its two Eco RI sites bypartial digestion with Eco RI, blunting with DNA polymerase I (Klenowfragment) and dNTPs, and religating. The resulting plasmid was digestedwith Bam HI and ligated with synthetic Bam HI-Eco RI adapters. Theresulting plasmid was designated Zem229R. Zem229R was digested with HindIII and Eco RI, and the 520 bp fragment containing the SV40 and MT-1promoters was removed. The large fragment of Zem229R was then joined tothe ˜1100 bp Hind III-Eco RI fragment of pDX, which contains the SV40promoter/enhancer, the adenovirus major late promoter, and a set ofsplicing signals to construct ZMB-4 (FIG. 3). The PC962 sequence wasisolated from PC229/962 as an Eco RI fragment, which was then insertedinto ZMB-4 to construct PC962/ZMB-4 (FIG. 3).

C. Construction of Hybrid Protein C Coding Sequence

The human-bovine protein C coding sequence was constructed by joining anEco RI-Sst II fragment of human light chain cDNA (from PC962/ZMB-4) to aTaq I-Eco RI fragment of bovine heavy chain cDNA using a syntheticlinker. The linker was constructed by annealing oligonucleotides ZC2228(5'GGCTCGT 3') and ZC2229 (5' CGCCGAGCAGC 3'). The hybrid proteinencoded by the resultant sequence has the amino acid sequence of humanprotein C through the first amino acid of the heavy chain followed bythe remainder of the bovine heavy chain: (H Pre-pro)-(HL-chain)-cleavage site (RRKR)-(H activation peptide)-Leu-(B H chain),where the sequence at the human-bovine junction is: ##STR13##

The hybrid cDNA was assembled by joining the cDNA fragments and linkerwith Eco RI-digested vector ZMB-3 in a four-part ligation. Expressionvector ZMB-3 was constructed from Zem228 (EP 319,944) and pDX (Hagen etal., U.S. Pat. No. 4,784,950, incorporated by reference herein). Zem228is a pUC18-based expression vector containing a unique Bam HI site forinsertion of foreign DNA between the mouse metallothionein-I promoterand SV40 transcription terminator. Zem228 also contains an expressionunit comprising the SV40 early promoter, the neomycin resistance gene,and the SV40 terminator. Thus, in Zem228 the inserted gene is under thecontrol of the metallothionein-l promoter and SV40 terminator, and thevector can be selected with the antibiotic neomycin. Zem228 was modifiedto delete its two Eco RI sites by partial digestion with Eco RI,blunting with DNA polymerase I (Klenow fragment) and dNTPs, religation,digestion with Bam HI and ligation with Bam HI-Eco RI adapters toconstruct plasmid Zem228R. Zem228R was digested with Hind III and EcoRI, and the 520 bp fragment containing the SV40 and MT-1 promoters wasremoved. The large fragment of Zem228R was then joined to the ˜1100 bpHind III-Eco RI fragment of pDX, which contains the SV40promoter/enhancer, the adenovirus major late promoter, and a set ofsplicing signals. The resultant vector was designated ZMB-3 (FIG. 4).

The ZMB-3 vector containing the hybrid human-bovine protein C codingsequence was transfected into tk⁻ ts13 BHK cells (ATCC CRL 1632).Transfectants were selected in Dulbecco's modified Eagle's Medium (DMEM)containing 10% fetal bovine serum and 500 μg/ml G-418. Conditioned mediawere harvested, and the recombinant protein C was purified byimmunoaffinity chromatography on a PCL-2-Sepharose column. This columnwas prepared by coupling a monoclonal antibody (designated PCL-2)specific for the Ca⁺⁺ -bound light chain of protein C to CNBr-activatedSepharose (Pharmacia, Piscataway, N.J.).

The samples were applied to the column in the presence of 10 mM CaCl₂.The column was washed with 50 mM Tris HCl, 1.0M NaCl, 10 mM CaCl₂, pH7.5. Protein C was eluted from the column with 15 mM EDTA in 50 mMTris-HCl, pH 7.5.

EXAMPLE II Resistance of Hybrid Protein C to Inactivation

The ability of a protein C inhibitor, α-1-antitrypsin, to inhibitactivated bovine protein C (obtained from Enzyme Research Labs, SouthBend, Ind.) and immuno-affinity purified, activated recombinant humanprotein C (from baby hamster kidney cells transfected with pDX/PC962)was compared with the inhibition of the activated human-bovine protein Chybrid. To activate the protein C molecules, each was combined withprotein C activator from Agkistrodon contortrix contortrix (ACC-Cobtained from W. Kisiel, Univ. of New Mexico; see, Kisiel et al., J.Biol. Chem, 262:12607-12613 (1987)).

To assay resistance to inactivation, a 200 μl solution of each protein(50 μg/ml in TBS 50 mM Tris pH 7.5, 150 mM NaCl!+15 mM EDTA) wascombined with 60 ng ACC-C and 5 μl BSA (50 mg/ml). The mixtures wereincubated at 37° C. for 90 minutes. A 20 μl sample of each activatedprotein C was combined with 5 μl BSA (50 mg/ml) and 0, 20, 40, or 80 μlof 1 mg/ml α-1-antitrypsin (Sigma Chemical Company, St. Louis, Mo.) inTBS to a final reaction volume of 105 μl. The mixtures were incubated at37 C for 181/2 hours, then 20 μl of each sample was combined with 80 μlof 1 mM chromogenic substrate (#336 Spectrozyme PCa, obtained fromAmerican Diagnostica) and incubated at room temperature forapproximately ten minutes. Color development was measured at 405 nm.

The results, shown in FIG. 5, indicate that while activated humanprotein C was readily inactivated by α-1-antitrypsin, both the bovineand bovine-human hybrid were resistant to inactivation.

EXAMPLE III Resistance of Hybrid Protein C to Inactivation by HumanPlasma

The inactivation of bovine-human protein C and human protein C (PC962)by human plasma was examined. The experiments were performedsubstantially as outlined in Example II, with the followingmodifications.

The bovine-human hybrid protein C and PC962 human protein C wereactivated by incubating 7.5 μg of each, or a BSA control, with 375 ngACC-C in 100 μl of TBS/BSA for 90 minutes at 37°. Twenty microlitersamples of activated protein C were added to wells of 96-well microtiterplates, then 0, 5, 10 or 20 μl of citrated human plasma was added toeach well. The sample volumes were adjusted to 100 μl with TBS/BSA, andthe plates were incubated overnight (16 hours) at 37° C. The assays weredeveloped by removing 20 μl (in duplicate) from each sample and addingthem to 80 μl chromogenic substrate (0.75 mM). The absorbance at 405 nmwas determined after incubating for about ten minutes at roomtemperature.

The results, shown in FIG. 6, indicate that the activity of humanprotein C decreased much more rapidly than that of the hybrid protein Cwhen the proteins were exposed to human plasma. The activated hybridprotein C appeared to have about three times greater chromogenicactivity than the activated human protein C in the absence of plasma.The hybrid protein also appeared to have a half-life about four timesgreater than the human protein.

The rates of inactivation of hybrid and human protein C molecules byhuman plasma were then compared. The assays were performed substantiallyas described above, using 7.5 μg of PC962 or hybrid protein C activatedovernight (18 hours) with 37.5 ng ACC-C in 100 μl TBS/BSA at 37° C. A 10μl sample from each was withdrawn and added to 190 μl TBS/BSA. Themixtures were placed on ice, then 250 μl citrated human plasma was addedto each, and the assays were incubated at 37° C.

Samples (20 μl) were withdrawn at 0, 105, 185 and 240 minutes and addedto 80 μl chromogenic substrate, and the absorbance at 405 nm determinedas above.

The results, shown in FIG. 7, suggest that the half-life of the hybridprotein C in human plasma was about three times that of the humanactivated protein C. The hybrid protein and human activated protein Chad approximately equivalent anticoagulant activity in human plasma.This suggests that the bovine-human hybrid protein C will beparticularly useful in treating humans, in that lower or less frequentdoses of protein C will need to be administered to a patient, therebydecreasing the cost and inconvenience of therapy to the patient.

EXAMPLE IV Anticoagulant Activity of Hybrid Protein C

The anticoagulant activity of the activated hybrid protein C molecule ofExample I was compared to that of native human APC in the APTT assay.Two micrograms of isolated hybrid protein C or recombinant PC962 werecombined with 50 ng ACC-C in 100 μl of TBS. The mixtures were incubatedat 37° C. for one hour, then combined with 100 μl of normal human plasmaand 100 μl Actin FS (Dade, Miami, Fla.). The resulting mixtures wereincubated at 37° C. for 100 seconds, then 100 μl of activated protein Cin TBS was added. After an additional 100 seconds at 37° C., 100 μl of1M CaCl₂ was added to each sample, and the clotting times were measured.Results, shown in Table II, indicated that the hybrid protein C hasanticoagulant activity in human plasma comparable to that of the nativehuman protein.

                  TABLE II                                                        ______________________________________                                                    Clotting Time (Seconds)                                           APC (ng)      PC962     Hybrid                                                ______________________________________                                         0            51        51                                                    20            64.6      62.8                                                  40            73.6      69.2                                                  60            83.6      76.7                                                  80            84.9      82.3                                                  100           88.4      90.3                                                  ______________________________________                                    

EXAMPLE V Bovine Sequence Substitutions Into Human Heavy Chain

A. Substitutions in Zymogen Protein C

To produce a protein C molecule with a substantially human heavy chainand having increased stability and increased half-life in human plasmawhen compared to authentic human activated protein C, sequences ofbovine protein C heavy chain are substituted for corresponding sequencesof the human heavy chain.

One modification of the human heavy chain involves the substitution ofbovine heavy chain amino acids Gln-Glu-Ala-Gly-Trp (amino acids 19-23;numbering according to Foster et al., Proc. Natl. Acad. Sci. USA 81:4766-4770 (1984), incorporated herein by reference and as shown in FIG.8) for amino acids Lys-Met-Thr-Arg-Arg in the human heavy chain (aminoacids 17-21; numbering according to Foster et al., id. and as shown inFIG. 8). To encode the substituted amino acids, site specificmutagenesis was employed with the synthetic oligonucleotide ZC2451 (5'CTC ATT GAT GGG CAG GAG GCT GGA TGG GGA GAC AGC CC 3'). The protein CSst I fragment of pDX/PC962 (Example I) was cloned into vector M13mp10(Messing, Meth. Enzymol. 101:20-77 (1983), incorporated herein byreference). Single-stranded template DNA was prepared as above andsubjected to site directed mutagenesis using the oligonucleotide ZC2451,essentially as described by Zoller and Smith, DNA 3:479-488 (1984),incorporated herein by reference, using the two primer method witholigonucleotide ZC550. Positive clones were selected and sequenced toconfirm the mutagenesis. The mutagenized sequence was recovered fromreplicative form DNA as a Pst I-Sst I fragment. This fragment wasjoined, in a four-part ligation, with a -592 bp Eco RI-Pst I fragmentfrom plasmid PC962/ZMB-4 (comprising the 5' protein C coding sequence),a ˜700 bp Sst I-Eco RI fragment from plasmid PC962/ZMB-4 (comprising the3' protein C coding sequence), and Eco RI-digested and phosphatasedZMB-4. Plasmids were screened for correct insert orientation byrestriction enzyme digestion. A correct plasmid was selected and used totransfect tk⁻ ts13 BHK cells (ATCC CRL 1632) by calcium phosphateco-precipitation, as described in Example I. Transfectants producingprotein C were selected at 500 nM methotrexate at 2-3 dayspost-transfection, and cell-conditioned media were prepared. Themodified protein C was purified from the culture supernatant asdescribed above. The sensitivity of the modified protein C toinactivation by alpha-1-antitrypsin and human plasma factors was assayedas described below.

Another modification of the human heavy chain, made either separately orin conjunction with other substitutions described herein, is thesubstitution of bovine amino acids Arg-Asp-Glu-Thr (heavy chain residues148-151) for human heavy chain amino acidsHis-Ser-Ser-Arg-Glu-Lys-Glu-Ala (human heavy chain residues 146-153)using the synthetic oligonucleotide ZC2452 (5' GCT GGG GCT ACA GAG ACGAGA CCA AGA GAA ACC GC 3'). The Sst I-Eco RI fragment of pDX/PC962(Example I) was cloned into vector M13mp10. Single-stranded template DNAwas prepared and subjected to site directed mutagenesis with ZC2452using the two primer method as described above. Positive clones wereselected and sequenced to confirm the substitution. The mutagenized SstI-Eco RI fragment was then reisolated from phage replicative form DNAand joined, in a four-part ligation, to a ˜330 bp Eco RI-Sal I fragmentfrom plasmid PC962/ZMB-4 (comprising the 5' protein C sequence), a ˜730bp Sal I-Sst I fragment from plasmid PC962/ZMB-4 (comprising the middleportion of the protein C sequence), and Eco RI-digested and phosphatasedZMB-4. Plasmids were screened for correct insert orientation byrestriction enzyme digestion, and a correct construction was selected.Transfection was performed as described above and protein C containingthe modified site was harvested from conditioned medium of thesuccessful transfectants. The sensitivity of the heavy chain modifiedprotein C to inactivation is compared to that of unmodified protein C asdescribed.

A substitution of the bovine sequenceTyr-Asn-Ala-Cys-Val-His-Ala-Met-Glu-Asn-Lys (heavy chain amino acids169-179) for the human heavy chain sequenceHis-Asn-Glu-Cys-Ser-Glu-Val-Met-Ser-Asn-Met (human heavy chain residues171-181) was used to provide enhanced resistance of the protein Cmolecule to inactivation. The Sst I-Eco RI fragment of pDX/PC962 wascloned into vector M13mp10, single-stranded template DNA was preparedand then subjected to site directed mutagenesis with syntheticoligonucleotide ZC3044 (5' CCC GTG GTC CCG TAC AAT GCA TGT GTC CAT GCCATG GAA AAC AAG GTG TCT GAG AAC ATG CTG 3') using the two primer methodas described above. As above, positive clones were selected andsequenced to confirm mutagenesis. Replicative form (RF) DNA from onepositive clone was digested with Sst I and Eco RI, and the 700 bp bandwas recovered.

To construct the expression vector for the 3044 mutant, the 700 bpfragment was ligated with Eco RI-digested, calf intestinal alkalinephosphatase-treated Zem229R, the 335 bp Eco RI-Sal I protein C fragmentfrom PC229/962 and the 730 bp Sal I-Sst I protein C fragment fromPC229/962. A correct construction, termed PC3044/Zem229R, was identifiedby digestion with Bgl II, Eco RI, Pst I and Ava II.

The PC3044/Zem229R vector was transfected into BHK 570 cells. After twodays the cells were split into 1 μM methotrexate. After two weeks ofgrowth the cells were screened by immunofilter assay with a monoclonalantibody against the heavy chain of human protein C and aperoxidase-conjugated rabbit anti-mouse second antibody. Positive cloneswere detected using the ECL substrate (Amersham). Individual positiveclones were picked and expanded, and conditioned media were collected.The mutant protein was purified from the media using thecalcium-dependent monoclonal antibody PCL-2, and assayed as describedfurther below.

Amino acid residues 249-260 of the bovine protein C heavy chain(Lys-Ala-Gln-Glu-Ala-Pro-Leu-Glu-Ser-Gln-Pro-Val) are substituted usingsite directed mutagenesis for human heavy chain residues 251-262(Arg-Asp-Lys-Glu-Ala-Pro-Gln-Lys-Ser-Trp-Ala-Pro). The Sst I-Eco RIprotein C fragment of pDX/PC962 was cloned into vector M13mp10,single-stranded template DNA was prepared and subjected to site directedmutagenesis with synthetic oligonucleotide ZC2454 (5' GGG CAC ATC AAAGCT CAG GAG GCC CCT CTT GAG AGC CAG GTG CCT TAG CGA CCC 3') using thetwo primer method as described above. Positive clones were selected andsequenced to confirm mutagenesis, and the mutagenized Sst I-Eco RIfragment was reisolated from RF DNA. The mutagenized fragment was thenused to construct an expression vector for the zymogen form of proteinC. The vector was constructed by ligating the mutagenized RF fragment,the ˜592 bp Eco RI-Pst I fragment from PC962/ZMB-4, the ˜460 bp PstI-Sst I fragment from PC962/ZMB-4, and Eco RI-digested and phosphatasedZMB-4. The resulting vector was then used to transfect tk⁻ ts13 BHKcells as described above, and the cells were selected in 1 μMmethotrexate. Successful transfectants were identified and cultured, andprotein was purified from cell-conditioned media and assayed forresistance to a-1-antitrypsin. The results indicated that the 2454mutant construct did not show increased resistance to theα-1-antitrypsin compared to human activated protein C.

B. Protein Characterization

Human protein C (PC962), the human-bovine hybrid (LMH), 2451, 2452 and3044 were tested for resistance to α-1-antitrypsin and human plasma, asgenerally described above. The proteins were incubated at 37° C. for 3hours in ACC-C (using a ratio of 100:1 protein C:ACC-C) to activatethem. Protein concentrations were adjusted to give approximately equalchromogenic activities (standardized to PC962).

Resistance to a α-1-antitrypsin (AAT) was determined using 140 μlreaction volumes in TBS (pH 7.4) containing 140 μg/ml BSA and 800 ngactivated protein C and from 0 to 80 μg ATT. The mixtures were incubatedfor 16 hours at 37° C., then 20 μl samples were removed from each tubeand added to 80 μl of 1 mM Spectrozyme PCa (American Diagnostica) inmicrotiter plates. After about 20 minutes the A₄₀₅ of the reactionmixtures was read. A plot of relative chromogenic activity vs.α-1-antitrypsin concentration is shown in FIG. 9. About three times asmuch α-1-antitrypsin was required to inhibit mutant 2451 by 50% ascompared to 962 (wild type human protein C), mutant 2452 and mutant3044. There was essentially no inhibition of the hybrid LMH.

Time courses for inactivation of the protein C mutants in human plasmawere determined and compared to wild type. Fifty microliters activatedprotein C (27 μg/ml) was added to 200 μl of pooled, citrated humanplasma. The mixtures were incubated at 37° C. Fifty microliter sampleswere removed and placed on ice at 0, 30, 75, 120 and 300 minutes. Twentymicroliters from each time point sample was transferred to microtiterwells with 80 μl of 1 mM Spectrozyme PCa. The A₄₀₅ was read afterseveral minutes. Results are shown in FIG. 10. PC2451 was similar to thehybrid LMH, being substantially more resistant to inactivation thanmutant 3044, which was more resistant than 2452 and wild type.

Time course experiments for inactivation in human plasma were repeatedfor hybrid LMH, wild type PC962 and mutant PC2451, with assays performedessentially as above but time points were taken at 0, 15, 30 and 60minutes, and the samples were immediately diluted into 60 μl ice-coldTBS containing 5 mM EDTA. Twenty microliters of 4 mM Spectrozyme PCa wasadded and the A₄₀₅ was read after several minutes. The results, shown inFIG. 11, confirmed that both the mutant PC2451 and hybrid LMH weresubstantially more resistant to inactivation than the human wild typeprotein C.

C. Substitutions in Activated Protein C

A DNA sequence encoding an activated protein C precursor with thecleavage site sequence Arg-Arg-Lys-Arg was constructed by mutagenesis ofthe wild-type protein C sequence. The resultant sequence (designated1058) was analogous to that encoding PC962, but lacked the portionencoding the activation peptide. The amino acid sequence at the junctionbetween the light and heavy chains of the 1058 protein is presented inTable 1.

The protein C sequence present in plasmid p594 was altered in a singlemutagenesis to delete the codons for the activation peptide and insertthe Arg-Arg codons at the processing site. A mutagenesis was performedaccording to standard procedures on the 870 bp Sst I fragment from p594cloned into an M13 phage vector using oligonucleotides ZC1058 (5' CGCAGT CAC CTG AGA AGA AAA CGA CTC ATT GAT GGG 3') and ZC550 (5' TCC CAGTCA CGA CGT 3').

A DNA sequence encoding an activated protein C precursor having thelinker sequence Lys-Lys-Arg-Ala-Asn-Ser-Arg-Arg-Lys-Arg between thelight (amino acids 1-149) and heavy chains was constructed Thisconstruct was designated PC2274 (Table 1).

To construct the PC2274 sequence, the PC1058 Sst I fragment was insertedinto M13mp10 and mutagenized according to standard procedures with theoligonucleotide ZC2274 (5' GAG AAG AAG CGC GCC AAC TCC AGA AGA AAA CGACT 3'). The mutagenized RF DNA was digested with Pst I and Sst I and the˜430 bp fragment was recovered.

The activated protein C expression vector was constructed by ligatingthe ˜430 bp Pst I-Sst I fragment from the PC2274 RF, theZC2454-mutagenized Sst I-Eco RI fragment (Example V.A), the -592 bp EcoRI-Pst I fragment from PC962/ZMB-4, and Eco RI-digested and phosphatasedZMB-4. A vector having the desired insert orientation was identified byrestriction enzyme digestion and was used to transfect tk⁻ ts13 BHKcells as described above.

It is evident from the above results that compositions are providedhaving substantially the activity of human protein C while possessing aresistance to inactivation by a-1-antitrypsin and human plasma factors.These results are especially encouraging, in that modified protein Cmolecules may now be employed as therapeutic or prophylacticcompositions which have an increased stability in human plasma and,accordingly, an increased half-life when compared to preparations ofhuman protein C purified from plasma or produced by recombinant means.The efficacy, convenience and economics of lower dosages and lessfrequent administration are among the advantages conferred by thecompositions of the present invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A method of treating protein C deficiency in apatient, said method comprising administering to said patient aprophylactically or therapeutically effective dose of a protein Ccomposition comprising:a) protein C having a light chain and ahuman-like heavy chain; b) a recombinant zymogen protein C moleculecomprising a light chain and a human-like heavy chain, which heavy chaincontains one or more amino acid substitutions in the human protein Cheavy chain sequence of FIG. 8, wherein said protein C molecule whenactivated is capable of inactivating human plasma Factors Va and VIIIaand has increased resistance to inactivation by human plasma oralpha-1-antitrypsin when compared to naturally occurring activated humanprotein C; or c) a biologically active chimeric protein C moleculecomprising light and heavy chain polypeptides, wherein the light chainis substantially homologous to the human protein C light chain and theheavy chain is substantially homologous to a heavy chain of a protein Cmolecule from a mammal other than human.
 2. A method of promotingfibrinolysis in a patient comprising administering to said patient atherapeutically effective dose of a protein C composition comprising:a)protein C having a light chain and a human-like heavy chain; b) arecombinant zymogen protein C molecule comprising a light chain and ahuman-like heavy chain, which heavy chain contains one or more aminoacid substitutions in the human protein C heavy chain sequence of FIG.8, wherein said protein C molecule when activated is capable ofinactivating human plasma Factors Va and VIIIa and has increasedresistance to inactivation by human plasma or alpha-1-antitrypsin whencompared to naturally occurring activated human protein C; or c) abiologically active chimeric protein C molecule comprising light andheavy chain polypeptides, wherein the light chain is substantiallyhomologous to the human protein C light chain and the heavy chain issubstantially homologous to a heavy chain of a protein C molecule from amammal other than human.
 3. A method of treating or preventingthrombosis in a patient comprising administering to said patient aprophylactically or therapeutically effective dose of a protein Ccomposition comprising:a) Protein C having a light chain and ahuman-like heavy chain; b) a recombinant zymogen protein C Moleculecomprising a light chain and a human-like heavy chain, which heavy chaincontaining one or more amino acid substitutions in the human protein Cheavy chain sequence of FIG. 8, wherein said protein C molecule whenactivated is capable of inactivating human plasma Factors Va and VIIIaand has increased resistance to inactivation by human plasma oralpha-1-antitrypsin when compared to naturally occurring activated humanprotein C; or c) a biologically active chimeric protein C moleculecomprising light and heavy chain polypeptides wherein the light chain issubstantially homologous to the human protein C light chain and theheavy chain is substantially homologous to a heavy chain of a protein Cmolecule from a mammal other than human.
 4. A pharmaceutical compositioncomprising an effective amount of a protein C composition comprising:a)protein C having a light chain and a human-like heavy chain; b) arecombinant zymogen protein C molecule comprising a light chain and ahuman-like heavy chain, which heavy chain contains one or more aminoacid substitutions in the human protein C heavy chain sequence of FIG.8, wherein said protein C molecule when activated is capable ofinactivating human plasma Factors Va and VIIIa and has increasedresistance to inactivation by human plasma or alpha-1-antitrypsin whencompared to naturally occurring activated human protein C; or c) abiologically active chimeric protein C molecule comprising light andheavy chain polypeptides, wherein the light chain is substantiallyhomologous to the human protein C light chain and the heavy chain issubstantially homologous to a heavy chain of a protein C molecule from amammal other than human, and a physiologically acceptable carrier.